Occupational exposure to reproductive toxicants occurs via inhalation, skin absorption, or ingestion. Thousands of chemicals exist in the workplace; there is limited or no toxicological information including reproductive and developmental (R/D) toxicity available for many industrial chemicals. Toxicity testing on animals represents one of the largest uses of animals yet it also has been an extraordinarily challenging area in which to implement in vitro alternatives. Since the report Toxicity Testing in the 21st Century by the National Research Council (NRC, 2007) envisioned that in vivo animal testing can eventually be replaced by a combination of in silico and in vitro approaches, the demand to identify in vitro models for R/D toxicity has grown. In this proposal, we will examine and establish a mini-testis model from testicular cell lines, which will finally eliminate the need o animals. We will examine and optimize protocol for the establishment of mini- testis. We will establish a toxicity-based high throughput mini-testis model and develop an integrated pathway- based high content (HCA) and high throughput (HTP) screening assay for R/D toxicity evaluation. The Specific Aims are (1) To examine and optimize in vitro 3D-TCCS model from testicular cell lines; (2) To establish a toxicity-based high throughput 3D-TCCS model for R/D toxicity evaluation; (3) To develop an integrated pathway-based high content (HCA) and high throughput (HTP) screening assay in the 3D-TCCS model for R/D toxicity evaluation. The output of the project will be the establishment of a pathway-based in vitro mini-testis model. This model will provide cost-efficient screen tool for potential R/D toxicity of chemicals in the workplace, wll provide importance data for establishing exposure limit in workplace for effective prevention of R/D toxicity in workplace such as MANUFACTURING, MINING, OIL AND GAS EXTRACTION and PUBLIC SAFETY. The research proposed will result in publication, conference proceedings, and generate R/D toxicity data regarding the chemicals in workplace, and provide data for risk assessment. This funding opportunity will support the PI's career development in reproductive toxicology, his long-term effort in seeking and promoting in vitro animal alternatives. Therefore, this proposal meets the mission of Cancer, Reproductive, and Cardiovascular Research Program (CRC). Intermediate outcomes from our research is the development of in vitro models that monitor and that can identify potential for reproductive toxicity would provide important alternatives to in vivo testing and start to address the public's concern for screening more compounds for R/D toxicity. The ultimate goal of the research by providing a cost-efficient screen tool to identify R/D toxicants in the workplace will significantl improve the risk assessment of reproductive toxicity in the workplace. The identification of reproductive toxicants will reduce the risk of reproductive malfunction of hazardous exposures. Therefore, our proposed project is within the NIOSH CRC's Research to Practice (r2p) strategy to promote the transfer and translation of research findings into disease prevention, the objectives of Healthy People 2020.